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The Complementarity Determining Region 2 of BV8S2 (V8.2) Contributes to Antigen Recognition by Rat Invariant NKT Cell TCR¹

Elwira Py^2,*, Olga Naidenko, Sachiko Miyake, Takashi Yamamura, Ingolf Berberich^*, Susanna Cardell^3,, Mitchell Kronenberg and Thomas Herrmann^4,*

^* Institute for Virology and Immunobiology, Würzburg University, Germany; Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, San Diego, CA; Department of Immunology, National Institute of Neuroscience, Tokyo, Japan; and Immunology Section, Department of Cell and Molecular Biology, Lund University, Lund, Sweden

	Abstract

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Invariant NKT cells (iNKT cells) are characterized by a semi-invariant TCR comprising an invariant -chain paired with -chains with limited BV gene usage which are specific for complexes of CD1d and glycolipid Ags like -galactosylceramide (-GalCer). iNKT cells can be visualized with -GalCer-loaded CD1d tetramers, and the binding of mouse CD1d tetramers to mouse as well as to human iNKT cells suggests a high degree of conservation in recognition of glycolipid Ags between species. Surprisingly, mouse CD1d tetramers failed to stain a discrete cell population among F344/Crl rat liver lymphocytes, although comprised iNKT cells are indicated by IL-4 and IFN- secretion after -GalCer stimulation. The arising hypothesis that rat iNKT TCR recognizes -GalCer only if presented by syngeneic CD1d was then tested with the help of newly generated rat and mouse iNKT TCR-transduced cell lines. Cells expressing mouse iNKT TCR reacted to -GalCer presented by rat or mouse CD1d and efficiently bound -GalCer-loaded mouse CD1d tetramers. In contrast, cells expressing rat iNKT TCR responded only to -GalCer presented by syngeneic CD1d and bound mouse CD1d tetramers only poorly or not at all. Finally, CD1d-dependent -GalCer reactivity and binding of mouse CD1d tetramers was tested for cells expressing iNKT TCR comprising either rat or mouse AV14 (V14) -chains and wild-type or mutated BV8S2 (V8.2) -chains. The results confirmed the need of syngeneic CD1d as restriction element for rat iNKT TCR and identified the CDR2 of BV8S2 as an essential site for ligand recognition by iNKT TCR.

	Introduction

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The hallmark of invariant NKT cells (iNKT cells)⁵ is the expression of a TCR with characteristic invariant -chain rearrangement and limited BV (V) usage which recognizes glycolipids like -galactosylceramide (-GalCer) in a CD1d-restricted manner (1). Mouse iNKT TCR -chains rearrange the variable gene 14 (AV14) and joining gene 18 (AJ18), which pair with -chains of high CDR3 variability comprising BV8S2 and to lesser extent to BV7 or BV2 (2, 3, 4). The human iNKT TCR is composed of AV11/AJ18 (homolog of mouse AV14) -chains paired with BV11 (homolog of mouse BV8) -chains (2, 5). In the rat, homologous -chain rearrangements have also been found (6). A contribution of iNKT cells in the control of tumors, infections, and autoimmune diseases (reviewed in Refs. 7, 8, 9) has been demonstrated in many mouse models and by clinical observations in humans.

iNKT TCR ligands are endogenous or microbial glycolipids that are presented by the nonpolymorphic MHC class I-like molecule CD1d. Crystal structures of complexes of CD1d and glycolipid Ags have been reported very recently (10, 11, 12). Natural ligands are isoglobotrihexosylceramide (13) and -anomers of various glycosphingolipids, which have been isolated from ₄-proteobacteria (14, 15). Other ligands activate only small subpopulations of iNKT cells. Their features are reviewed in Ref. 16 . Still the most thoroughly characterized Ag of iNKT cells is the -anomer of galactosylceramide (-GalCer), which was originally isolated from a marine sponge. Essentially all iNKT cells respond to -GalCer, and they can be visualized with -GalCer-loaded CD1d oligomers (reviewed in Ref. 17). Of special importance to our study is the observation that -GalCer-loaded mouse CD1d oligomers bind to human iNKT cells (18, 19, 20) and human CD1d tetramers stain mouse iNKT cells. Thus, it appears that ligand recognition is highly conserved throughout evolution (21).

The rat also expresses genes for CD1d (22, 23) and the AV14, AJ18 (6), and BV8S2 (24) gene segments, which are highly similar to those of the mouse (>80% sequence similarity of the translated products). Peculiar to the rat is the existence of a multimember AV14 gene family and the organ-specific preferences of certain AV14AJ18 rearrangements. Within the BN/SsNHsd genome, 10 AV14 genes have been identified (25), and analysis of F344/Crl rearrangements identified five AV14 family members, which based on CDR2 sequence similarity, have been divided into the type I and type 2 genes. Rearrangements of type I genes (AV14S1, AV14S2, AV14S4 (a pseudogene); and AV14S8, described in this paper) have been reported to be predominant within intrahepatic lymphocytes (IHL), whereas rearrangements of the type II gene AV14S3 are more frequently found in spleen, bone marrow lymphocytes, and thymocytes (6, 25). In all cases, either a G or an A have been found at position 93, located at the VJ junction (6), which is similar to the mouse, where mostly a G but also rarely A, V, or I are found in this region (2, 26).

Despite this information on the genetics, knowledge of the phenotype, function, and Ag recognition by rat iNKT TCR-bearing cells is rather limited. The comparison of NKR-P1A-positive rat T lymphocytes (6) with mouse NKT cells has elucidated some differences in terms of phenotype and functions. First of all, NKR-P1A (rat homolog of mouse NK1.1)-positive T cells found in spleen and liver (6, 27, 28) were of CD8 phenotype and showed no preferential BV usage. This is in stark contrast to the mouse, in which most of the NK1.1-positive T cells, and nearly all iNKT cells, are CD4⁺ or CD4^–CD8^–. Secondly, NKR-P1A-positive rat T cells produce IFN- but not IL-4 upon in vitro CD3 stimulation (28). Thus, it appears that these cells are not the equivalent of mouse iNKT cells.

Nevertheless, there is also evidence that favors the existence of typical iNKT cells in the rat. Matsuura et al. (6) showed that coculture of F344/Crl IHL with CD1d-transduced hepatocytes leads to the accumulation of cells with AV14 transcripts, and they identified AV14AJ18 rearrangements in a NKR-P1A^high subset of intrahepatic T lymphocytes of LEC rats (29). Additionally, another group has reported the generation of CD4⁺ or CD4^–CD8^– NKR-P1A⁺ T cell clones from PVG rats that home to the liver and produce Th1 and Th2 cytokines (30). However, to our knowledge, -GalCer reactivity of rat T lymphocytes or binding of -GalCer-loaded CD1d oligomers have not been described yet. Both groups reported staining of the presumed rat iNKT cells by the BV8-specific mAb R78 (6, 30), which depending on the Tcrb haplotype binds to different rat homologs of mouse BV8S2. In F344/Crl rats (Tcrb^a haplotype), R78 reacts with BV8S4 (BV8S4A2) but not with BV8S2 (BV8S2A2), whereas in PVG rats (Tcrb^l haplotype) BV8S4 (BV8S4A1) is not functional and R78 Ab stains positively BV8S2 (BV8S2A1) (24, 31).

This paper describes our attempts to characterize F344/Crl rat iNKT cells, their phenotype, and their -GalCer reactivity. Although like mouse NKT cells, the lymphocytes isolated from F344/Crl rat liver secreted cytokines upon -GalCer in vitro stimulation, they could not bind -GalCer-loaded mouse CD1d tetramers (mouse CD1d tetramers). To test the species specificity of recognition of -GalCer-CD1d complexes, mouse and rat iNKT TCR were cloned, and a panel of cell lines expressing mouse and rat, wild-type or mutated iNKT TCR were generated. The AV14⁺ lines were tested for rat vs mouse CD1d-restricted -GalCer recognition and for binding of mouse CD1d tetramers. The results confirmed the hypothetical species specificity of CD1d-dependent -GalCer recognition by rat iNKT cells and allowed, for the first time, definition of the important role of the CDR2 as a germline-encoded TCR region for this recognition.

	Materials and Methods

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Animals

C57BL/6 mice and LEW/Crl rats were bred in the animal facilities of the Institute for Virology and Immunology, University of Würzburg, Würzburg, Germany. F344/Crl rats were obtained from Charles River Wiga. All animals were maintained under specific pathogen-free conditions and were used at 6–10 wk of age.

Cell preparation and culture

Mouse and rat IHL were isolated using discontinuous Percoll (Pharmacia Biotech) gradients (40%/70% or 40%/80%) as described in Ref. 32 . In both cases, liver was perfused with complete medium (via the portal vein) until it became opaque. Then the organ was homogenized by passing through a metal mesh, and cells were washed with medium. Cells were resuspended in 40% isotonic Percoll solution and underlaid with 70 or 80% isotonic Percoll solution. After 25 min of centrifugation at 900 x g at room temperature, mononuclear cells were isolated from the interface. Remaining erythrocytes were removed from the cell pellet by lysis with TAC buffer (Tris-ammonium chloride, 20 mM Tris (pH 7.2), 0.82% NH₄Cl). Thymocytes were isolated by passing the organ through a metal sieve followed by washing with complete medium. Primary cells and cell lines were cultured at 37°C with 5% CO₂ and H₂O-saturated atmosphere. Almost all cell types were cultured in RPMI 1640 (Invitrogen Life Technologies) supplemented with 5 or 10% FCS, 100 mM sodium pyruvate, 0.05% (w/v) glutamine, 10 mM nonessential amino acids, and 100 µM 2-ME (Invitrogen Life Technologies). DMEM with the same supplements was used for transfection of 293T cells, conducted to produce retroviruses for gene transfer.

Cloning and expression of rat and mouse iNKT TCR

Rat AV14S8 -chain was directly cloned from F344/Crl IHL cDNA, whereas the rat AV14S1 -chain construct was generated using molecular biology methods. For the F344/Crl AV14S8 -chain, RNA was isolated from cytoplasmic extracts of 10⁶ IHL following the protocol of the RNeasy MiniKit (Qiagen). The cDNA was synthesized according to the manufacturer’s RT-PCR protocol supplied with a First Strand cDNA Synthesis Kit (MBI Fermentas). PCR was performed with HotStar DNA polymerase (Qiagen), using AV14-specific primers: (MWG-Biotech) rV14/1,2,3-Fow (5'-TTT GGG GCT AGG CTT CTG-3'), RCend-STOP-Rev (5'-TCA ACT GGA CCA CAG CCT TAG CG-3'). PCR products were cloned into TOPO cloning vector (Topo pCR2.1-TOPO-TOPO TA Cloning Kit; Invitrogen Life Technologies) and sequenced using an ABI sequencer. Subsequently, rat AV14S8 -chain DNA was ligated into EcoRI sites of pczCGZ5 IEGZ retroviral vector.

An AV14S1 -chain with a V domain amino acid sequence described by Matsuura et al. (6) was generated using molecular biology methods. F344/Crl genomic DNA was amplified by PCR with rV14EcoRI-Fow (5'-GGG CTA GAA TTC TGC AGA AAA ACC ATG GGG AAG C-3') and r/mV14Rev (5'-CAC CAC ACA GAT GTA GGT GGC AG-3') primers. This DNA was digested with EcoRI and Esp enzymes and gel purified. The resulting fragment, which encoded the leader and the first 72 aa of mature V region peptide, was coligated with a EspI-F344/Crl cDNA-BamHI fragment (encoding the JC terminus of another liver-derived -chain) into the EcoRI/BamHI sites of pczCGZ5 IEGN vector, and the insert was sequenced. The generation of the rat BV8S2 -chain and its mutants has been described elsewhere (33).

A mouse type 1 NKT cell TCR was cloned by RT-PCR from mouse KT12 hybridoma (34) using -chain (mV14-EcoRI-Fow: 5'-GGG GAA TTC AAC CAT GAA AAA GCG CC-3'; and mC14-EcoRI-Rev: 5'-CCC GAA TTC CTC AAC TGG ACC ACA GCC-3') and -chain (mV8.2-BamHI: 5'-CGG GAT CCT GAG ATG GGC TCC AGG CTC TTC-3'; and mCend-BamHI: 5'-GGG GGA TCC TCA GGA ATT TTT TTT CTT GAC C-3')-specific primers. Mouse AV14S1A2 -chain DNA was ligated into the EcoRI site of pczCGZ5 IEGN (containing genes for neomycin resistance and enhanced green fluorescence protein), and mouse BV8S2 -chain DNA was ligated into BamHI sites of pczCGZ5 IEGZ (containing genes for zeozin resistance and enhanced green fluorescence protein) retroviral vectors (35).

Rat TCR -chains (AV14S8 and AV14S1) as well as mouse AV14SA2-TCR -chain were expressed together with C57BL/6 mouse or rat BV8S2 TCR -chains in BW58r/mCD28 cells using a transient three-plasmid expression system. BWr/mCD28 cells are BW58 TCR^– mouse hybridoma transduced with chimeric rat/mouse CD28 molecule (36). These cells are especially suitable for the analysis of Ag presentation by CD80-positive APC (33). Expression of transduced -chains was estimated from the green fluorescence of the reporter gene. Cell surface expression of transduced TCR was analyzed by staining with anti-mouse CD3 mAb. When necessary to obtain similar levels of TCR expression, cell lines were sorted using a FACSVantage (BD Biosciences) machine or by coculture in selection medium containing 1 mg/ml neomycin (Invitrogen Life Technologies) or 250 µg/ml zeozin (CAYLA), alternatively.

Cloning and expression of rat and mouse CD1d

P80rCD80 cells were transduced with mouse or rat CD1d. P80rCD80 cells are P80 cells (P815 mouse mastocytoma transduced with rat CD80; Ref. 37) which, to increase rat CD80 expression, were additionally infected with pczCGZ5IZ or pczCGZ5IEGZ retroviral vectors expressing genes for rat CD80 and zeozin resistance. These have been generated by RT-PCR from the CD80-containing BCMGSC vector (37) and subsequently cloned into the EcoRI sites of both retroviral vectors. P80rCD80 cells transduced with rat CD1d are designated as P80rCD80rCD1d, those transduced with mouse CD1d as P80rCD80mCD1d.

Mouse CD1d was cloned from A20mCD1d cell line (38) by RT-PCR using the following primers: mCD1d-EcoRI-Fow (5'-GGG GAG AAT TCC GGC GCT ATG CGG TAC CTA CC-3'); and mCD1d-EcoRI-Rev (5'-GGT GGA ATT CAG AGT CAC CGG ATG TCT TGA TAA G-3'). The sequence of the insert showed a complete overlap with the mouse CD1d sequence available in the gene bank under X13170 (39). Rat CD1d cDNA was obtained by RT-PCR using RNA isolated from F344/Crl rat bone marrow as a template and CD1d-specific primers: N366 (5'-TCG GAG CCC AGG GCT GTG TAG A-3'); and rCD1dRev (5'-TTC TGA GCA GAC AAG GAC TGA-3'). PCR product was cloned into TOPO cloning vector and sequenced. The sequence was identical with rat CD1d (GenBank accession number AB029486) published by Katabami et al. (23). Mouse and rat CD1d DNA were cloned into EcoRI site of pczCGZ5IZ and pczCGZ5 IEGZ vectors, respectively, and were further used for retroviral infection of P80rCD80 cells.

The expression of mouse CD1d was tested with the CD1d-specific mAb 1B1 (BD Pharmingen), whereas expression of rat CD1d was assessed from the green fluorescence of the EGFP reporter gene. Surface expression of rat CD1d was also confirmed with a novel rat CD1d-specific mAb (E. Pyz and T. Herrmann, unpublished observations). The Ag-presenting cell lines were enriched for CD1d expression by cell sorting or selection with antibiotics.

Stimulation with -GalCer in vitro

-GalCer was generated as described (40). The reactivity of mouse and rat IHLs to -GalCer was tested by culture of IHL (1 x 10⁵ cells/well of a 96-well round-bottom plate) in the presence of -GalCer (100 ng/ml), vehicle (DMSO), or complete medium for 24 h at 37°C. The level of IL-4 and IFN- released into culture supernatants was determined using ELISA kits (BD Pharmingen).

To analyze the -GalCer reactivity of TCR-transduced cell lines, mouse and rat thymocytes (1 x 10⁶ cells/well), or CD1d-transduced cells (P80mCD1drCD80, P80rCD1drCD80, 5 x 10⁴ cell/well) used as APC were loaded with either -GalCer (1–100 ng/ml) or vehicle (DMSO) for 1–2 h before the addition of responder cells. As a positive control, TCR-positive cell lines were stimulated with plate-bound anti-mouse CD3 mAb 145C11. After 24 h of culture, supernatants were taken, and the secreted mouse IL-2 was quantified using a commercial ELISA Kit (BD Pharmingen).

Immunofluorescence and flow cytometry

For the staining, 2 x 10⁵ cells were diluted in 100 µl of FACS buffer (PBS (pH7.4), 0.1% BSA, 0.02% NaN₃) and were treated for 10 min at 4°C with normal mouse Ig (Sigma-Aldrich) or mouse FcR-specific 2.4G2 Ab to block unspecific binding or binding to Fc receptors. Subsequently, cells were stained for 30 min with labeled mAbs, washed, and stained with another mAb or analyzed with a FACScan or FACSCalibur flow cytometer (BD Biosciences).

All mouse and rat mAbs were obtained from BD Pharmingen and are given with their clone names: mouse V8.1, 8.2, 8.3 (F23.1); mouse CD3 -chain (145-C11); mouse CD1d (1B1); mouse CD4 (GK1.5); mouse CD8 (53-6.7); NK1.1 (PK136); BV8S4A1 and BV8S4A2, V8.2 of LEW rats and V8.4 of F344/Crl rat (R78); rat TCR -chain (R73); rat CD4 (W3/25); rat CD4 (OX35); rat CD8 (3.4.1.); rat NKR-P1A (10-78). Abs were usually FITC or PE labeled. Biotinylated mAbs, when used, were visualized with streptavidin-CyChrome. Unconjugated Abs, used in indirect immunofluorescent staining, were detected by using fluorochrome-conjugated Abs: PE- or Cy5.5-conjugated (Fab')₂ fragment of donkey anti-mouse IgG or goat anti-hamster IgG FITC obtained from Dianova or Serotec).

Staining with -GalCer-loaded mouse CD1d-PE tetramer

-GalCer-loaded or control mouse CD1d-PE tetramers were generated as described in Ref. 18 . Tetramer staining of mouse/rat IHL- or TCR-transduced cell lines was performed as normal FACS staining, but with incubation for 1 h at room temperature. Tetramer concentrations were 350 (high tetramer) or 35 ng/50 µl cell suspension (low tetramer).

	Results

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Phenotype and -GalCer response of rat IHL

In mouse and human, the highest proportion iNKT cells can be found among intrahepatic lymphocytes. In an attempt to identify the corresponding population in rat, IHL of F344/Crl rats and C57BL/6 mice were compared for cell surface phenotype (Fig. 1A), binding of -Gal-loaded mouse CD1d tetramers (Fig. 1B), and -GalCer-induced cytokine production (Fig. 1C). In agreement with published data, about one-third of mouse IHL coexpressed NK1.1 (mouse homolog to rat NKR-P1A) and TCR. More than 20% of IHL coexpressed NK1.1 and CD4, but very few coexpressed NK1.1 and CD8. As shown in Fig. 1B, 27% of IHL show costaining of -GalCer-loaded CD1d tetramer and anti-CD3, with the tetramer positive cells having a lower or intermediate level of expression of CD3. 25.5% of IHL were costained by tetramer and anti-NK1.1 (data not shown) and 20.4% by tetramer and anti-CD4, whereas only very few (0.38%) stained with CD1d tetramer and CD8-specific mAb (data not shown).

View larger version (51K): [in this window] [in a new window]   FIGURE 1. Phenotypic and functional analysis of typical iNKT cell features of C57BL/6 mouse and F344/Crl rat IHL. A, Two-color flow cytometry for coexpression of NK1.1 or NKR-P1A and indicated T cell markers. Percent of positive cells are indicated by numbers in the upper right quadrant. B, Two-color flow cytometry for binding of -GalCer-loaded or unloaded mouse CD1d tetramers to CD3+ positive (upper right quadrant) or CD3– (upper left quadrant) C57BL/6 mouse or F344/Crl rat IHL. Percentages of tetramer-positive cells are given in the respective quadrants. C, IFN- or IL-4 secretion during 24-h stimulation of 1 x 105 rat or mouse IHL with 100 ng/ml -GalCer dissolved in DMSO, vehicle (DMSO alone), or medium alone. Ordinate, Cytokine concentration in picograms per milliliter.

To test whether the lack of binding of mouse CD1d tetramer to rat IHL was due to the absence of -GalCer-specific cells in F344/Crl rat liver, the -GalCer reactivity of F344/Crl and C57BL/6 IHL (Fig. 1C) was compared. After 24 h of stimulation with -GalCer (100 ng/ml), mouse and rat liver lymphocytes produced both IFN- and IL-4. The amount of rat IL-4 reached 15% of that secreted by mouse cells. The IFN- production by rat IHL exceeded that of mouse IHL, but rat IHL showed also a high level of background IFN- production.

The -GalCer-induced activation of cytokine production in conjunction with the detection of AV14AJ18 rearrangements in rat IHL strongly support the existence of an iNKT cell population in F344/Crl rats, although these cells could not be detected by mouse CD1d tetramer. This could be a consequence of 1) an extremely low frequency of rat iNKT cells and/or 2) a requirement for presentation of -GalCer by syngeneic CD1d (species specificity), which finally would result in a lack of binding of mouse CD1d tetramers to rat iNKT TCR. To test the latter hypothesis, iNKT TCRs were cloned and expressed in TCR-negative BW58r/mCD28 cells and tested for mouse CD1d tetramer binding. In addition, these lines as well as lines expressing iNKT TCR variants were tested for reactivity to -GalCer presented by mouse or rat CD1d.

Cloning and transduction of mouse and rat CD1d and iNKT TCR

Cloning, transduction, and quantification of surface expression of iNKT TCR was performed as described in Materials and Methods. Three AV14 -chains were cloned into a retroviral vector carrying an EGFP as reporter gene. Two of them comprised V-encoded amino acid sequences identical with that of rat AV14S1 and rat AV14S8. The mouse AV14S1A2-chain was cloned from the -GalCer-reactive mouse C57BL/6-derived iNKT cell hybridoma KT12. All AV14 -chains were coexpressed with different mouse or rat BV8S2 -chains, the properties of which will be discussed later in this section.

The sequences of the tested iNKT TCR -chains are compared in the upper part of Fig. 2. Both rat AV14S1 and rat AV14S8 -chain comprise type 1 AV14 sequences. The V domain of the rat AV14S1 -chain is identical with sequences previously found by Matsuura et al. in F344/Crl rat liver (6). The rat AV14S8 -chain sequence was directly cloned from F344/Crl IHL, as described in Materials and Methods. AV14S8 has not yet been described for F344/Crl rats, but an identical sequence has been found in the BN/SsNHsd rat genome, where it has been named AV14S8 (25). A peculiarity of the AV14S8-comprising -chain used in this study may be the valine located at position 93 of the VJ junction which corresponds to the adult type of AV14AJ18 rearrangements (26). Otherwise, the mature V domains of the two rat TCRs differed by the following substitutions: K1R, Q15E, and K51T.

View larger version (23K): [in this window] [in a new window]   FIGURE 2. Alignment of amino acid sequences of the mature peptides TCR chain proteins (-chain and -chain) and CD1d molecules used or discussed in this study. Underlined parts of the TCR sequences indicate localization of CDRs. Parts of CD1d sequences in italics indicate -helical regions. Amino acid sequences were deduced from the nucleotide sequences, the accession numbers of which can be found in GenBank: rAV14S8 -chain, DQ340291; rAV14S1 -chain, DQ340293; mAV14S1, AY158221; mAV14S1A2 (KT12 hybridoma), DQ340292; BV8S2A1 TCR35/1 -chain, AY228549. Mutants entry indicates localization of the CDR2 and CDR4/HV4 substitutions introduced in the TCR35/1 -chain, which are highlighted by bold letters. mBV8S2 TCR KT12, DQ340294; mCD1d (mouse CD1d), X13170.1; rCD1d (rat CD1d), AB029486.

The lower part of Fig. 2 presents the amino acid sequence of the -1 and -2 domains of rat and mouse CD1d. The -helical parts of CD1d are marked. The helices of the 1 domains differ in 3 aa. Visualization of the of the PDB files 1ZHN (10) and 1Z5L (12) of the mouse CD1d crystal structure by Swiss-PDB-viewer (http://swissmodel.expasy.org/SM_TOPPAGE.html) shows that T74 points upwards and K81 outwards, defining them as theoretical contact sites with the TCR. I83 points into the binding groove. The -helical parts of the -2 domain differ by 7 aa. With exception of the R157S, side chains of all substitutions show upwards and provide possible contacts for the TCR. In contrast to the differences in potential TCR contacts, those amino acids shown to provide H bonds with -GalCer are conserved (12). Both CD1d genes were expressed in P815 cells (P80rCD80) overexpressing rat CD80 as described in Materials and Methods.

Species specificity of CD1d restriction in Ag recognition by rat iNKT TCR

First, we tested three responder cell lines for their -GalCer reactivity and their capacity to bind mouse CD1d tetramers. The lines were BWr/mCD28 cells expressing: 1) as positive control, mouse iNKT TCR isolated from the KT12 hybridoma which consisted of a mouse AV14S1A2 -chain and mouse BV8S2 -chain; 2) rat AV14S1 -chain with the CDR2+4 -chain mutant; 3) the rat AV14S8 -chain with the same -chain mutant. The BV8S4-like CDR2+4 -chain mutant was used, because there is circumstantial evidence that in F344/Crl rats, iNKT cells express the BV8S4-comprising -chains (6). The two rat TCR lines expressed very similar levels of TCR, whereas expression of the mouse TCR was considerably lower (Fig. 3). Cell lines were tested three to five times for their -GalCer-induced IL-2 secretion. IL-2 levels after CD3 ligation were quite similar, with the exception of sometimes considerably lower IL-2 production by the mouse iNKT TCR-transduced line (data not shown). Fig. 3 shows data from one representative assay of -GalCer-induced IL-2 secretion. The APC-type thymocytes vs CD1d-transduced P80 cells and the origin of the transduced CD1d (rat vs mouse) considerably affected the outcome of the assay. Generally, with CD1d-transduced P80rCD80 cells as APC, Il-2 production was much higher than with thymocytes. This may reflect the differences in the level of CD1d and CD80 surface expression in primary vs CD1d-transduced cells (data not shown). In assays with mouse thymocytes as APC, some background IL-2 production was found, even if TCR-negative BW58 cells were used as responders, suggesting that IL-2 was secreted by -GalCer-stimulated thymocytes (Figs. 3 and 5). With regard to a possible species specificity in CD1d-restricted -GalCer recognition, all three lines responded to -GalCer presented by rat CD1d-expressing cells, whereas -GalCer mouse CD1d complexes stimulated only mouse iNKT TCR responder cells. In addition, the stimulation of the line expressing the rat AV14S1 -chain was considerably stronger than of the rat AV14S8 -chain-expressing line.

View larger version (42K): [in this window] [in a new window]   FIGURE 3. A, Species specificity of CD1d-restricted -GalCer recognition by rat iNKT TCR-transduced cells. The graphs in the upper row indicate degree of IL-2 production (please note the different scales of the ordinates) by TCR-transduced BW58r/mCD28 cells after stimulation with -GalCer presented by different APC-expressing mouse or rat CD1d. Transduced TCR: , vector control; , mAV14S1A2+mBV8S2 (mouse -chain + mouse -chain); , AV14S8 + rat CDR2+4 -chain (rat AVS8 -chain + BV8S4-like rat -chain); •, AV14S1 + rat CDR2+4 -chain (rat AVS1 -chain + BV8S4-like rat -chain). Amino acid sequences of the TCR chains used by these are given in Fig. 2. The type of -GalCer-presenting cells and concentrations of -GalCer used for stimulation are indicated on top of the respective graphs and at the abscissa, respectively. Zero ng/ml indicates the use of vehicle control. B, Upper row, CD3 expression of TCR-transduced cell lines used in A. Binding of isotype control () or anti-CD3 (). Lower row, Mouse CD1d tetramer staining. Binding of unloaded control (350 ng/50 µl sample, ) and of -GalCer-loaded tetramers (350 ng/50-µl sample, ). The type of transduced TCR is given on top of the histograms. The numbers in the histogram give ratios of MFIs for staining with -GalCer-loaded mouse CD1d tetramers divided by that for staining with anti-CD3.

View larger version (25K): [in this window] [in a new window]   FIGURE 5. Analysis of mouse CD1d restricted -GalCer recognition by chimeric mouse - rat--chain iNKT TCR reveals contribution of CDR2 to ligand recognition by iNKT TCR. Shown is IL-2 production of BW58r/m CD28 transduced with AV14S1A2 -chains and various -chains indicated by the symbols in the graph to -GalCer presented by indicated APC. BW indicates cells transduced with vector control. Every section of the columns indicates IL-2 production by cells expressing a certain --chain combination. Note the variation of the scales indicating IL-2 production in the various graphs. -GalCer concentrations are given in ng/ml. Vehicle designates culture with solvent (DMSO) only.

Effects of iNKT TCR - and -chain differences on CD1d-restricted -GalCer recognition

We have previously analyzed the effects of CDR2 and/or CDR4 mutations of rat BV8S2 on the recognition of peptide Ags and superantigens (33). To learn whether the known BV encoded (super)Ag recognition sites may also contribute to -GalCer recognition, AV14 -chains were coexpressed with the various rat BV8S2 -chain mutants and a mouse BV8S2 -chain. These lines were then tested for the response to -GalCer presented either by rat or mouse CD1d and for binding of -GalCer-loaded mouse CD1d tetramer.

All lines expressed similar levels of TCR (summarized in Fig. 6) and produced similar amounts of IL-2 after stimulation with anti-CD3 mAb, with the exception of the mouse -chain-expressing lines, which sometimes showed a rather low level of IL-2 production (data not shown). All lines were tested two to five times; and although the overall degree of stimulation varied between experiments, the patterns of -GalCer reactivity remained the same. Figs. 4 and 5 show results from a representative experiment comparing all cell lines and Fig. 6 summarizes the results of all experiments.

View larger version (29K): [in this window] [in a new window]   FIGURE 6. Survey of -GalCer responsiveness of TCR-transduced cell lines, their CD1d tetramer binding, and TCR expression. The left part of the figure summarizes functional data of three to five experiments on the response to -GalCer presented by rat or mouse CD1d by cell lines expressing the indicated iNKT TCR combinations (see also Figs. 3–5). The central part gives an estimate on CD1d tetramer binding for different cell lines. The stacked bars depict the ratio of MFIs obtained by staining with 350 ng (tetramer high) or 35 ng (tetramer low) of CD1d tetramer divided by the MFI of anti-CD3 staining. Numbers on the abscissa indicate this ratio. Examples for the staining are given in Fig. 3. The right part gives the MFI of CD3 staining of the cell lines used to generate of the data depicted in the central part of the graph.

View larger version (23K): [in this window] [in a new window]   FIGURE 4. CD1d-restricted -GalCer recognition of rat iNKT TCR. Species specificity of CD1d restriction. Shown is the -GalCer-induced IL-2 production of BW58r/m CD28 transduced with rat AV14S1 or AV148 -chains and various -chains and different types of APC. Each section of the column indicates IL-2 production by cells expressing a certain --chain combination. The -chain is indicated at the top of the graph, the -chains are indicated by the symbols in the graph. BW, Cells transduced with vector control. Ordinate, Type of APCs and the origin of CD1d. Note the variation of the scales indicating IL-2 production in the various graphs. -GalCer concentrations are given in nanograms per milliliter. Vehicle designates culture with solvent (DMSO) only.

In contrast to the variation in the response to -GalCer presented by mouse CD1d, recognition of rat CD1d--GalCer complexes was largely unaffected by the -chain of iNKT TCR. All lines expressing TCR comprising the rat or mouse AV14S1 -chains (Figs. 4 and 5) showed a very similar response. The somewhat lower IL-2 production of the line coexpressing rat AV14S1 -chain and the CDR4 mutant -chain probably reflects a generally weaker capacity in TCR-triggered IL-2 production, because anti-CD3 induced IL-2 secretion (not shown) was only about one-half of that found for the other lines. Less clear were the results for cell lines expressing rat AV14S8 -chain. In two of four experiments, -chain composition affected the response to rat CD1--GalCer complexes of the lines. An example for such a differential response is given in Fig. 4, where the lines expressing the CDR2 or CDR2+4 mutant -chains reacted far better than those lines expressing the wild-type BV8S2 or the CDR4 mutant.

Finally, effects of the -chain composition were also seen for the three mouse -chain-expressing lines. The rat AV14S8 -chain/mouse -chain-expressing line completely lacked -GalCer reactivity (Fig. 4), whereas the rat AV14S1 -chain/mouse -chain expressing line responded to -GalCer if it was presented by rat CD1d-transduced P80rCD80 cells (Fig. 4), implicating rat V interactions with CD1d in imparting the observed species specificity. Only the mouse AV14S1A2 -chain/mouse -chain expressing line responded irrespective of the types of APC or origin of CD1d used to present the -GalCer (Fig. 4).

Differential binding of -GalCer-loaded mouse CD1d tetramers to iNKT-TCR-transduced lines

All cell lines were also tested for TCR expression and binding of -GalCer-loaded tetramers at two different concentrations. In all cases, binding of unloaded tetramer control was negligible. Fig. 6 summarizes data from such an experiment and gives an overview of the results obtained in the functional assays. The capacity to bind -GalCer-loaded mouse CD1d tetramers is presented by the ratio of MFI of tetramer binding and MFI of anti-CD3 binding. The best binding was found for the TCR comprising mouse AV14S1 -chain paired with the CDR2, CDR2+4 mutants of rat BV8S2 -chains or the mouse BV8S2 -chain (Figs. 3 and 6), which is consistent with their exclusive capacity to respond to -GalCer presented by mouse CD1d. At least 8 times weaker was the tetramer binding of lines coexpressing mouse -chains and rat BV8S2 and CDR4 mutants.

Interestingly, the tetramer binding varied also between the rat -chain-expressing lines. The poorly responding rat AV14S8 -chain-expressing lines showed essentially no binding, whereas at least some tetramer binding was found for the more reactive lines coexpressing rat AV14S1 -chain and the suitable CDR2 or CDR2+4 mutated -chains. Finally, and again consistent with the functional assays, the rat -chains paired with mouse -chain bound no tetramer, whereas the original mouse iNKT TCR bound it very well. Indeed, the efficient binding of this TCR at the lower tetramer concentration suggests a rather high avidity of the original mouse iNKT TCR for -GalCer-mouse CD1d complexes, consistent with measurements conducted with other mouse iNKT cell hybridomas and T cell populations.

Fig. 6 summarizes our results on the CD1d-restricted -GalCer response and binding of -GalCer-loaded mouse CD1d tetramers to iNKT TCR-transduced cell lines. It appears that the lack of reactivity to -GalCer presented by mouse CD1d results from an impaired binding of the rat iNKT TCR - rather than -chain to mouse CD1d. Furthermore, comparison of iNKT TCR sharing the same -chain but comprising different -chains revealed that the amino acid composition of CDR2 of the -chain strongly affects the CD1d-restricted glycolipid reactivity.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
This study was initiated to characterize the phenotype and the -GalCer response of rat iNKT cells in a side by side comparison of mouse and rat IHL. As previously described (1, 6, 28), 30% of mouse IHL coexpressed NK1.1 and TCR and were either CD4⁺ or CD4^–CD8^–, whereas rat IHL comprised rather low numbers of NKR-P1A (rat homolog of NK1.1) and TCR⁺ cells, most of them being CD8⁺. Our attempts to directly detect rat iNKT cells by staining with -GalCer-loaded mouse CD1d tetramers failed, although the capacity of rat IHL to produce IFN- and IL-4 production after stimulation with -GalCer suggested that there is indeed a functional iNKT cell population in F344/Crl rats. Analysis of newly generated cell lines expressing CD1d and iNKT TCR of both species allowed us to directly demonstrate the functionality of the rat elements of cognate Ag recognition by iNKT cells. In addition, this analysis revealed that Ag recognition by rat iNKT TCR required its presentation by syngeneic CD1d, which was unexpected, given that mouse and human CD1d tetramers and dimers (18, 19, 20) bind to iNKT cells of the opposite species. Nevertheless, despite this cross-species reactivity, mouse iNKT TCRs bind mouse CD1d better than human CD1d, as was shown with -GalCer-loaded mouse CD1d dimers (4). In addition, the weakly binding human dimers showed a stronger preference for mouse BV8S2 iNKT TCR than for mouse dimers, a result that underlines the substantial contribution of the -chain to binding of -GalCer CD1d complexes (4).

Interestingly, mouse iNKT TCR-transduced lines responded quite well to -GalCer presented by rat and by mouse CD1d, whereas the rat iNKT TCR-expressing lines responded only when Ag was presented by rat CD1d. What could be the reason for the need of syngenicity between iNKT TCR and CD1d only in one direction? One possibility could be that higher numbers of -GalCer complexes on rat CD1d⁺ APCs could have compensated for the generally low avidity of rat iNKT TCR for CD1d, in particular for mouse CD1d. This possibility cannot be formally excluded but seems to be rather unlikely because homologous types of APC were used for presentation. Alternatively, we suggest a higher degree of promiscuity either in Ag recognition by mouse vs rat iNKT TCR or in Ag presentation by rat vs mouse CD1d.

With the help of chimeric and mutated iNKT TCR, we could identify TCR regions, which contribute to binding of -GalCer and (mouse) CD1d. Cell lines expressing TCR comprising a mouse iNKT TCR -chain and a suitable -chain transgressed the threshold for the induction of a response to Ags presented by mouse CD1d, and these cells efficiently bound mouse CD1d tetramers. The differential reactivity of the rat BV8S2 -chain mutants allowed us for the first time to demonstrate the important role of BV-encoded parts in the -GalCer response, without a possible interference by CDR3 diversity. In addition, analysis of mutants swapping the CDR2 of BV8S2 with that of BV8S4 provided evidence for an involvement of the CDR2 of the -chain in recognition of the -GalCer-CD1d complex. In this context, it is of interest that the CDR2 of rat BV8S4, which in the combination with the mouse iNKT TCR -chain permitted binding of -GalCer-mouse CD1d complexes, and the CDR2 of mouse BV8S differ from each other by only one amino acid (Fig. 2). In contrast, the CDR2 of rat BV8S2, which in the interspecies comparison was nonpermissive, differed from that of mouse BV8S2 by 3 aa.

Rat Tcrb haplotypes vary in expression of functional BV8S2 and BV8S4 genes. The Tcrb^a haplotype, which is found in F344/Crl and DA rats, expresses BV8S2 and BV8S4, whereas the Tcrb^l haplotype of LEW/Cr, BN, and PVG rats expresses only BV8S2 (24, 31, 41, 42). These rat strains are widely used as models for autoimmune diseases; therefore, it is of special interest to investigate whether differences in reactivity to natural iNKT TCR ligands based on differences in the CDR2s of BV8S2 vs BV8S4 could lead to a rat strain-specific variation in iNKT T cell development or Ag reactivity.

The three -chains tested contributed not only to restricted recognition of syngeneic CD1d, but also to the overall magnitude of the -GalCer response. The lines expressing TCR with rat AV14S1 chains and mouse AV14S1A2 -chains showed a much better response than the rat AV14S8-expressing lines. By analogy to what is known from MHC-restricted recognition of peptide Ags, the differences in -GalCer reactivity of the two rat -chains could have been explained by the K50T substitution in the CDR2 and by the A93V difference in the CDR3 (43). Two reasons lead us to assume that the CDR2 difference is of minor importance. In a comprehensive study on a mouse AV14S1 polymorphism, Sim et al. (44) demonstrated that a pronounced CDR2 difference between mouse AV14S1A1 and AV14S1A2 (see also Fig. 2) had little if any effect on TCR-binding to -GalCer-CD1d complexes (44). Also, our own preliminary results (E. Pyz, I. Müller, and T. Herrmann, unpublished observations) obtained with rat AV14S1 and AV14S8 chain mutants showed little effect of the K50T substitution on the -GalCer response, whereas a pronounced effect was found for the V93A substitution.

To sum up, we showed that efficient activation of rat iNKT TCR-expressing lines requires presentation of -GalCer by syngeneic CD1d, and that reactivity to complexes of -GalCer and mouse CD1d can be obtained by replacing the rat -chain against that of the mouse and by using a -chain comprising the CDR2 of rat BV8S4.

This finding thus provides the first description of a germline-encoded CDR involved in ligand recognition by iNKT TCR. The generation and functional analysis of further chimeric rat/mouse iNKT TCR and of chimeric rat/mouse CD1d molecules should strongly facilitate the characterization of the TCR/CD1d/Ag complex. At a certain point of chimerism of TCR or CD1d, cells expressing iNKT TCR comprising rat/mouse -chain chimeras would be expected to gain specificity for -GalCer presented by mouse CD1d and mouse/rat CD1d chimeras should gain the capacity to efficiently present Ag to rat iNKT TCR. Finally, combined functional assays with cells expressing such chimeric or mutated receptors and ligands, at best together with binding studies of recombinant molecules, may even allow definition of direct contacts in the ternary complex comprising iNKT TCR/Ag and CD1d.

	Acknowledgments

 
We thank Kathrin Krejci and Ingrid Müller for excellent technical assistance and Niklas Beyersdorf and Barabara Sullivan for critical reading of the manuscript.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by European Graduate College "Gene regulation in and by microbial pathogens" (to E.P. and T.H.) and by National Institutes of Health Grant AI 45053 (to M.K.).

² Current address: Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, Wernher and Beit Building, South Groote Schuur Campus, Observatory, 7925, Cape Town, South Africa.

³ Current address: Department of Microbiology and Immunology, Goteborg University, Box 435, SE-405 30 Goteborg, Sweden.

⁴ Address correspondence and reprint requests to Dr. Thomas Herrmann, Institut für Virologie und Immunbiologie, Versbacherstrasse 7, 97078 Würzburg, Germany. E-mail address: herrmann-t{at}vim.uni-wuerzburg.de

⁵ Abbreviations used in this paper: iNKT cells, invariant NKT cells; -GalCer, -galactosylceramide; IHL, intrahepatic lymphocytes; MFI, mean fluorescence intensity.

Received for publication January 3, 2006. Accepted for publication March 10, 2006.

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